JP2007174937A - Particle carrying biological substance, sensor using the same and method for detecting specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a particle which can carry biological substances in larger amounts than those of conventional particles and can solve a problem that enough biological substances could not have been carried in porous particles. <P>SOLUTION: This rod-like particle is characterized by having meso-pores crossing the longitudinal direction of the rod-like particle and carrying a biological substance in the meso-pores. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a technique for immobilizing a biological material and stabilizing the biological material. More specifically, biosensors can be applied to particles that carry biological substances in mesopores.

  It is known that an enzyme easily loses its original function due to changes in the three-dimensional structure of the protein constituting the enzyme depending on heat and the environment. Various methods have been studied in order to stably handle enzymes and proteins. One of them is a method of carrying an enzyme or protein on a solid surface, for example, a technique that has been put into practical use, such as an immobilized enzyme.

For immobilization of enzymes, silica, fused quartz, porous inorganic materials, porous organic polymer materials and the like prepared by a sol-gel method are used. In recent years, there has been proposed immobilization of mesoporous materials, particularly mesoporous silica, formed using a surfactant molecular aggregate as a template. For example, Patent Literature 1 and Patent Literature 2 describe the technology.
JP 2000-139459 A JP 2002-95471 A

  However, there are some problems in the technology for immobilizing an enzyme on conventional mesoporous silica. That is, in the case of mesoporous silica such as MCM-41 and SBA-15 disclosed in Patent Document 1 and Patent Document 2, the pore diameter is small, and the protein that can be carried in the pore is small in size. It was limited. In addition, since mesoporous silica such as SBA-15 has a tube-shaped pore diameter formed in parallel to the long axis direction of the rod-shaped particles, the aspect ratio of the tube-shaped pores is large, and the inside of the pores. The diffusion of proteins and substrates was not always good. In addition, the number of pore openings on the surface was small, and the amount of protein introduced was small.

  The present invention has been made in view of the above problems, and uses mesoporous silica having mesopores with a small aspect ratio as a carrier for immobilizing biological materials. As a result of diligent investigations, the present inventors have used conventional mesoporous silica by utilizing such a mesopore structure having a small aspect ratio that intersects the major axis direction of rod-shaped particles as a carrier for immobilizing biological materials. In comparison, a large amount of biological material was adsorbed inside the mesopores. Furthermore, it was confirmed that the biological material was stabilized even after immobilization.

That is, the present invention
Rod-shaped particles,
Having mesopores intersecting the rod-shaped major axis direction;
The present invention provides particles that carry biological substances in the mesopores.

The present invention also provides
A sensor for detecting a specimen,
Rod-shaped particles having mesopores intersecting with the rod-shaped major axis direction, and particles carrying a biological material in the mesopores,
With electrodes,
A sensor for detecting an electrical output signal based on a reaction between the specimen and the biological substance carried in the mesopores is provided.

Furthermore, the present invention provides
In the specimen detection method,
A rod-shaped particle, having a mesopore intersecting with the rod-shaped major axis direction, and preparing a sensor using particles carrying a biological substance in the mesopore;
Applying a fluid containing a specimen to the sensor;
The present invention provides a specimen detection method comprising a step of detecting an output signal based on a reaction between the biological substance and the specimen.

  According to the present invention, it is possible to provide a particle having a small aspect ratio and capable of immobilizing a large amount of biological material in a mesopore. Thereby, it becomes possible to increase the detection sensitivity when applied to a biosensor or the like.

  Hereinafter, the present invention will be described in detail.

  First, the porous material used in the present invention will be described.

  FIG. 1 is a schematic view of particles in which mesopores used in the present invention intersect with the long axis direction of rod-shaped particles. In this specification, although the position of the mesopore is arranged at a position intersecting with the major axis direction of the rod-shaped particle or is described as being oriented in the minor axis direction of the rod-shaped particle, It represents the structure shown in FIG. 1, and does not represent another.

  Tubular mesopores are arranged in the short axis direction of the rod-like structure. As shown in FIG. 1, the tubular mesopores are typically packed with honeycombs. However, in the porous body used in the present invention, the tubular mesopores may be formed almost in parallel with the minor axis direction of the rod, and any arrangement may be used. For example, a structure in which meso holes are arranged at intersections of the meshes may be used.

  The rod-like structure in this specification refers to, for example, that shown in FIG. 1 and has a major axis and a minor axis. When confirmed with an electron microscope, the short axis is in the range of 150 nm to 500 nm, and the long axis is in the range of 0.5 μm to 2 μm. As shown in FIG. 1, the shape may be an ellipse, a rectangle, a rhombus, or a polygon.

  The mesopore (or mesopore) in the present invention is defined by IUPAC and refers to a pore having a pore diameter in the range of 2 nm to 50 nm. More preferably, the mesopore diameter is 10 nm to 30 nm. This is considered to be because the enzyme can be more stably supported by the interaction between the wall surface and the enzyme. Further, the length of the mesopores is preferably greater than 10 times and 30 times or less with respect to the pore diameter. This is because conventional mesopores have a long length, and sufficient enzymes cannot be supported. On the other hand, the particles of the present invention have a small aspect ratio and can carry a much larger amount of enzyme than conventional mesoporous particles.

  The mesopores of the mesoporous particles are formed by surfactant molecule aggregates (micelles). Under certain conditions, the same number of pores are formed because the number of associations of the molecules forming the micelles is equal. Various shapes such as a spherical shape, a tube shape, and a layer shape are known, but the shape of the micelle forming the mesoporous material according to the present invention is basically a tube shape. The tubes may be connected or separated.

  In the mesoporous material used in the present invention, any material that forms the pore wall of the porous material can be applied as long as it has the above-described pore structure. For example, there are titanium oxide, tin oxide, silicon oxide, and the like. Among them, a material containing silicon as a component is preferable, and silica is particularly preferably used. An organic silica hybrid material comprising an organic group containing one or more carbon atoms, two or more silicon atoms bonded to the organic group at two or more locations, and one or more oxygen atoms bonded to the silicon atom. But it ’s okay.

  A method for producing a mesoporous material having a small aspect ratio using a surfactant micelle as a template is described in, for example, Journal of the American Chemical Society, Vol. 126, p. 7740. However, the mesoporous material used in the present invention is not limited to these methods as long as it satisfies the characteristics of the mesoporous material.

  Hereinafter, a method for synthesizing short-axis-oriented mesoporous silica using a sol-gel method will be described.

  The reaction solution is a solution containing a surfactant, an organic molecule, and a substance that becomes a raw material of a target material such as a metal alkoxide. Depending on the material forming the pore walls, an appropriate amount of acid or the like as a hydrolysis reaction catalyst may be added.

  Depending on the target material, a halide, chalcogenide, metal alkoxide or the like is used as a raw material. For example, when the pore wall is silica, tetraethoxysilane or tetramethoxysilane which is a metal alkoxide is preferably used. Of course, silica sources other than alkoxides can be applied to the present invention.

  As the surfactant to be used, a nonionic surfactant such as a block copolymer containing polyethylene oxide as a hydrophilic group is used. However, usable surfactants are not limited to these, and are not particularly limited as long as the desired structure can be obtained.

  The orientation control of the tubular mesopores in the short axis direction is controlled by the organic molecules to be added and the amount of the organic molecules to be added. For example, rod-shaped mesoporous silica having a pore structure oriented in the minor axis direction is synthesized by adding n-decane.

  Common acids such as hydrochloric acid and nitric acid can also be used.

  The mesoporous material of the present invention can be synthesized by reacting the above reaction solution under hydrothermal conditions. The temperature at the time of synthesis is selected in the temperature range of 80 ° C to 150 ° C. The reaction time is about several hours to several days, and the reaction temperature and reaction time are optimized as appropriate.

  The mesoporous material synthesized in this manner is washed with pure water and then naturally dried in air, whereby an inorganic-organic composite powder material containing surfactant micelles as templates in pores is obtained. By removing the surfactant micelles of the template from the inorganic-organic composite powder material produced as described above, a short-axis-oriented mesoporous material with a small aspect ratio that can be used in the present invention is produced. Can do. There are various methods for removing the surfactant, and any method may be used as long as the surfactant can be removed without destroying the pore structure.

  The most commonly used method is a method of firing in an atmosphere containing oxygen. For example, by baking the synthesized material in air at 550 ° C. for 10 hours, the surfactant can be completely removed with almost no destruction of the mesopore structure. The firing temperature and time are preferably optimized depending on the material forming the pore walls and the surfactant used.

  About the mesoporous powder sample synthesize | combined by such a method, nitrogen gas adsorption / desorption measurement can be performed and the knowledge regarding a pore diameter can be obtained. The mesoporous material of the present invention is characterized in that the pore diameter is a substantially uniform diameter. As used herein, the uniform pore size refers to the pore size distribution determined by the Berret-Joyner-Halenda (BJH) method based on the results of nitrogen gas adsorption measurement, and the obtained pore size distribution is a single maximum value. Have Furthermore, in the pore size distribution, it indicates that 60% or more of mesopores in all mesopores are included in a range having a width of 10 nm. The pore diameter can be changed by appropriately selecting a surfactant described later.

  The periodic structure of the pores can be obtained by X-ray diffraction (XRD) measurement. The mesoporous material in the present invention is characterized by having at least one diffraction peak in an angular region corresponding to a structure period of 1 nanometer or more in XRD measurement.

  On the surface of the mesoporous material of the mesoporous material used in the present invention, it is possible to form a material different from the pore wall component, for example, a layer of organic matter or metal oxide.

  As a method of modifying the pore surface with an organic substance, a method of modifying the pore surface with a silane coupling agent is generally used. As a modification method using an inorganic oxide, a method of modifying the pore surface using an aqueous solution of a metal salt containing a metal capable of forming an oxide is generally used.

A silane coupling agent is a compound generally represented by a chemical formula of R—Si—X ₃ and has two or more different functional groups in the molecule. Said X is a site | part which can react with the porous body surface which consists of inorganic materials. For example, when the mesoporous material is silica, it is described in Sol-Gel Science 1989, page 662. Here, it is disclosed that hydrogen of a silanol group existing on the pore surface is replaced by an organosilicon group to form a Si—O—Si—R bond and form a layer of organic matter R on the pore surface. Yes.

  As X, a chloro group, an alkoxy group, an acetoxy group, an isopropenoxy group, an amino group and the like are known, but there is no particular limitation in the present invention. Further, as long as it can react with the pore surface and form an R layer, a bifunctional or monofunctional coupling agent may be used even if X is not a trifunctional group.

  On the other hand, R is an organic group, and an amino group, a carboxylic group, a maleimide group, or the like is preferably used, but is not limited to the organic group.

  Next, a method for modifying the pore surface using an aqueous solution of a metal salt containing a metal capable of forming an oxide will be described. The oxide here refers to a compound having at least one bond between an element such as a metal and oxygen. Examples of the metal that can form the oxide layer include titanium, aluminum, zirconium, and tin. For example, by treating mesoporous silica with an aqueous solution of zirconium oxynitrate, an oxide layer of zirconium can be formed on the surface. However, the metal constituting the metal salt is not limited thereto as long as it is an aqueous solution of a metal salt capable of forming a metal oxide layer on the pore surface.

  Subsequently, the biological material-immobilized material of the present invention will be described.

  An example of the structure of the material on which the biological substance is immobilized is schematically shown in FIG. In this figure, 21 is the structure having the orientation in the short axis direction, and 22 is a pore wall of the mesoporous material. Reference numeral 23 denotes biological materials such as proteins and enzymes, and reference numeral 24 denotes a substrate having specific binding properties with these biological materials and a fragment having reaction specificity.

  Further, although not shown, there may be an anchor that connects 22 and 23. This anchor may give an effect of suppressing and maintaining a large structural change of the biological material, but is not an essential component.

  As a component constituting the anchor, basically the same structure as that of the mesoporous material is desirable.

In particular, it preferably has the following functional groups for binding to biological materials.
Hydroxyl group, amide group, amino group, pyridine group, urea group, urethane group, carboxylic group, phenol group, azo group, hydroxyl group, maleimide group, silane derivative, aminoalkylene group One or more in each pore Contains biological material. Accordingly, the pores need to have a size suitable for immobilizing the biological material, and the size is appropriately determined depending on the size of the biological material to be immobilized.

  When immobilizing a biological substance in the pores, it is preferable to adsorb it on the surface of the pores by electrostatic bonding. However, it is possible to hold biological materials in the pores not only by electrostatic bonding but also by van der Waals force, hydrogen bonding, ionic bonding non-covalent bonding, and the like.

  Examples of biological substances to be immobilized include antigens, antibodies, proteins, and enzyme molecules. Further, it may be a fragment containing an active site such as Fab antibody. The biological material may be extracted from animals or plants or microorganisms, and may be cleaved if desired, or may be synthesized genetically or chemically.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to the content of an Example.

  In this example, mesoporous silica having substantially uniform tubular mesopores formed in parallel to the minor axis direction of the rod was produced and used as an enzyme immobilizing agent.

2.40 g of triblock copolymer (EO ₂₀ PO ₇₀ EO ₂₀ ; HO (CH ₂ CH ₂ O) ₂₀ (CH ₂ CH (CH ₃ ) O) ₇₀ (CH ₂ CH ₂ O) _{20 as a} nonionic surfactant H) was dissolved in 76.5 ml of pure water. In addition, 7.5 ml of 36 wt. % Concentrated hydrochloric acid was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, 13.9 g of n-decane was added and stirred at room temperature for 2 hours. Further, 0.027 g of NH ₄ F as a hydrolysis catalyst, and a material obtained by adding 5.10g of tetraethoxysilane (TEOS) was a precursor solution to the mixed solution. The composition (molar ratio) of the final precursor solution was TEOS: HCl: EO ₂₀ PO ₇₀ EO ₂₀ : NH ₄ F: n-decane: H ₂ O = 0.25: 0.9: 0.004: 0. 007: 1: 42.9.

  This precursor solution was stirred at 40 ° C. for 20 hours and reacted at 100 ° C. for 48 hours. The obtained white precipitate was sufficiently washed with pure water and vacuum-dried.

  The obtained powder sample was fired in air at 550 ° C., and the surfactant was decomposed and removed from the pores to form hollow pores. Removal of organic substances such as surfactants was confirmed by infrared absorption spectrum.

  As a result of evaluating the synthesized mesoporous silica powder by the X-ray diffraction method, as shown in FIG. 3, the diffraction peak attributed to the (100) plane of the hexagonal structure having a surface interval of 11.7 nm was started, (110), (200) , A diffraction peak attributed to the (210) plane was confirmed. This result shows that the pore structure of this mesoporous silica has a hexagonal arrangement with high regularity.

As a result of the nitrogen adsorption / desorption isotherm measurement at 77K, the adsorption isotherm shape became type IV in the IUPAC classification. B. E. T.A. The specific surface area calculated by the method was 700 m ² / g, and the pore volume was 1.88 ml / g. Further, when the pore diameter is calculated by the BJH method from the results of the adsorption isotherm, the pore diameter distribution of the mesoporous silica synthesized in this example is a narrow distribution having a single peak at 14.3 nm, Within 90% was within the distribution of 10 nm.

  Next, when observation was performed using a scanning electron microscope (SEM), a rod-shaped structure was confirmed as shown in FIG. Further, when SEM observation was performed at a high magnification, tubular mesopores having a diameter of 14 nm were oriented in the minor axis direction of the structure as shown in FIG. Moreover, in the cross-sectional view, as shown in FIG. 6, a relatively uniform tube-shaped mesopore formed a pore structure in which honeycomb packing was performed. During observation, the mesopore structure was not destroyed by the electron beam.

  Next, a horseradish peroxidase (abbreviated as HRP, average diameter = 4.8 nm, isoelectric point (IEP) = 7.8), which is a kind of oxidoreductase, in the mesopores of the synthesized mesoporous silica. After immobilization, the stability against heat and organic solvent was measured.

  HRP was adjusted to 5 mg / ml using 5 mM phosphate buffer (pH = 7.0), and 10 mg of the synthesized mesoporous silica was added to 1 ml of this enzyme solution. The mixed solution was stirred using a shaker at 4 ° C. for 20 hours to adsorb HRP into the mesoporous silica pores. After completion of the reaction, the mixture was centrifuged at 20000 g for 10 minutes at 4 ° C., and the precipitate of HRP-mesoporous silica was washed 3 times with pure water. At this time, each of the original enzyme solution and the supernatant was subjected to UV-Vis absorbance measurement. Using the absorption maximum at 403 nm of HRP, the adsorption amount of HRP to mesoporous silica was calculated from the concentration change before and after adsorption. The enzyme-immobilized mesoporous silica after washing was freeze-dried for 10 hours to obtain a powder sample. The adsorbed HRP showed a high value of 252 mg / g with respect to the weight of 1 g of mesoporous silica. Moreover, the adsorption amount of HRP changed by changing the pH of the phosphate buffer. From this result, it is considered that HRP and mesoporous silica pores are immobilized by electrostatic interaction.

  The introduction of HRP molecules into the pores was confirmed by a decrease in the amount of adsorption into the pores by measuring nitrogen adsorption on mesoporous silica after HRP adsorption.

  Next, the mesoporous silica immobilizing HRP produced by the above method was used to measure the resistance to an organic solvent by an oxidation reaction in toluene and the stability to heat by an oxidation reaction of phenol.

  In order to evaluate the enzymatic activity of HRP immobilized on mesoporous silica in an organic solvent, the oxidation reaction of 1,2-diaminobenzene in toluene using tert-butyl hydroperoxide as an oxidizing agent was used. . 8 ml of anhydrous toluene containing 50 mM 1,2-diaminobenzene was mixed with 2 ml of a 1.1 M tert-butyl hydroperoxide solution prepared by dissolving tert-butyl hydroperoxide in n-decane. 10 mg of HRP-immobilized mesoporous silica prepared by the above method was added to 1 ml of this mixed solution, and the reaction was started at 37 ° C. The absorbance at 470 nm of 1,2-dinitrobenzene produced by oxidation of 1,2-diaminobenzene was measured, and the enzyme activity of HRP in a toluene solvent was determined by determining the change with time.

  As a comparative test, 0.5 mg of HRP itself was prepared and subjected to the above oxidation reaction, and the increase in absorbance at 470 nm was similarly measured. The result is shown in FIG. Oxidation reaction in toluene did not occur at all with HRP (Free HRP) alone, but mesoporous silica with HRP immobilized showed very high activity. This is considered to be because HRP was denatured immediately after adding HRP to toluene, and it was found that high stability was expressed by immobilizing HRP on mesoporous silica.

  Subsequently, mesoporous silica with HRP immobilized and normal HRP without immobilization were heat-treated at 70 ° C. for 0 to 2 hours in a phosphate buffer solution. Then, the result of having measured the residual enzyme activity is shown in FIG. The thermal stability of HRP immobilized in mesoporous silica was determined by measuring the oxidative degradation rate of phenol. For the determination of phenol, a 4-aminoantipyrine colorimetric method was used.

  400 μl of 50 mM sodium acetate buffer (pH = 4.0) was added to 10 mg of HRP-immobilized mesoporous silica prepared by the above HRP adsorption, and heated at 70 ° C. for 30, 60, 90, and 120 minutes, respectively. Centrifugation was performed after heating, and the HRP-immobilized silica was washed twice with pure water. Next, 400 μl of 50 mM Tris-HCl (hydroxymethylaminomethane hydrochloride) buffer (pH = 7.5), 8 μl of 5000 ppm phenol aqueous solution, and 2 μl of 30% hydrogen peroxide water were added, and the mixture was reacted at 37 ° C. for 30 minutes. . After centrifugation, the supernatant was added with 150 μl and 1 M glycine aqueous solution (pH = 9.6) of 1% hexacyanoferrate and 1% 4-aminoantipyrine 150 μl and 300 μl, respectively, and stirred. Absorbance was measured.

  Non-immobilized HRP had its enzyme activity halved by heat treatment at 70 ° C. for 30 minutes, and showed only about 10% of the initial enzyme activity after 2 hours. On the other hand, in the mesoporous silica in which HRP is fixed, a high stabilization effect against heat was confirmed. Even after heat treatment at 70 ° C. for 2 hours, the enzyme activity was 90% or more.

  The results of measuring the temperature dependency of HRP using the above-described phenol oxidation reaction are shown in FIG. HRP and immobilized HRP were pretreated at 25 to 100 ° C. for 30 minutes, respectively, and then enzyme activity was measured. Normal HRP had a residual activity of 0% under conditions of 70 ° C. and 30 minutes, whereas HRP immobilized on a short-axis oriented mesoporous showed an activity of 50% or more even under the same conditions. Moreover, even if it processed at 100 degreeC, it did not become 0% and the enzyme activity of 40% was hold | maintained.

(Comparative Example 1)
As a comparative example, SBA-15 having a tubular pore diameter formed parallel to the long axis direction of rod-shaped particles was synthesized, and the adsorption effect of HRP and the stability effect in an organic solvent were measured. A method for synthesizing SBA-15 is described, for example, in Science 279, page 548.

As a result of evaluating the synthesized SBA-15 by the X-ray diffraction method, a diffraction peak attributed to the (100) plane of a hexagonal structure having a plane spacing of 9.8 nm was confirmed. SBA-15 synthesized from nitrogen adsorption isotherm measurement had a specific surface area of 800 m ² / g and a pore diameter of 7.4 nm.

  SRP-15 synthesized was subjected to the same HRP adsorption experiment as in Example 1. The amount of HRP adsorbed was 25 mg / g, showing an extremely small amount of adsorption of 1/10 or less compared to the short-axis oriented mesoporous silica used in the present invention. Moreover, from the nitrogen adsorption isotherm analysis using the sample after HRP adsorption, there is no decrease in the pore volume of SBA-15 before and after the adsorption of HRP, and HRP is hardly adsorbed in the SBA-15 pores. I understood that. Considering that the average diameter of HRP is 4.8 nm and the pore diameter of SBA-15 is 7.4 nm, it is expected that the adsorption was difficult due to the small number of pore openings. As a result of measuring the amount of HRP adsorbed on mesoporous silica over time, the time to reach the saturated adsorption amount was very short in the short-axis oriented mesoporous silica.

  Subsequently, the organic solvent stability of HRP was measured by the above method using this SBA-15, and compared with the mesoporous silica according to the present invention. Slight enzyme activity by HRP immobilized on SBA-15 was observed. However, in the SBA-15-immobilized HRP, 1,2-dinitrobenzene as a reaction product was gradually confirmed as the reaction started. In contrast, in the mesoporous silica of the present invention, 1,2-dinitrobenzene more than 10 times that of SBA-15 was confirmed immediately after the start of the reaction.

  Therefore, the present inventors have found that these results show that the SBA-15 has a large aspect ratio of the tubular pores because the tubular pore diameter is formed parallel to the long axis direction of the rod-shaped particles. As a result, the diffusion of HRP and the substrate from the outside to the inside of the pore and from the inside to the outside of the pore is poor, and the number of pore openings on the surface is small, so the introduction of HRP and the substrate is slow. I guessed it was the cause. From this result, it was revealed that the short-axis oriented mesoporous silica used in the present invention is excellent as a carrier for biological substances and the like.

  In this example, the short-axis oriented mesoporous silica surface prepared in Example 1 was modified with zirconium oxide to immobilize bovine serum albumin (abbreviated as BSA, diameter = 7.0 nm, IEP = 4.7). This is an example of measuring heat stability.

  10 g of zirconium oxynitrate dihydrate was added to 90 ml of pure water and dissolved at room temperature to prepare a 10 wt% zirconium oxynitrate aqueous solution. The short-axis oriented mesoporous silica synthesized in Example 1 was added to this solution and immersed for 20 hours. Thereafter, the supernatant was removed by centrifugation, washed 3 times with pure water, and dried at room temperature.

  As a result of evaluating the short-axis oriented mesoporous silica modified with zirconium by the X-ray diffraction method, the diffraction pattern showed almost the same as before modification, and it was confirmed that the periodic structure of the mesopores was not broken. Moreover, as a result of measuring the chemical bonding state on the silica surface using X-ray photoelectron spectroscopy (XPS), a peak due to Zr-O was confirmed, and that a zirconium oxide layer was formed on the silica surface. confirmed.

  Subsequently, BSA was immobilized in the pores of the mesoporous silica after the modification with zirconium, and the stabilizing effect was measured using an antigen-antibody reaction with an HRP-labeled anti-BSA antibody.

  5 ml of a solution prepared by adjusting BSA to 1 mg / ml using 10 mM phosphate buffer (pH = 5.0) was added to 10 mg of short-axis oriented mesoporous silica after zirconium modification, and the mixture was stirred at 4 ° C. for 6 hours. . As a result, BSA was immobilized in the mesopores of mesoporous silica. Then, it washed 3 times with pure water. In addition, when a BSA adsorption experiment was performed using mesoporous silica with short axis orientation not modified with zirconium, no adsorption was observed.

  An anti-BSA antibody (abbreviated as HRP-antiBSA) labeled with horseradish peroxidase was added to the BSA-immobilized mesoporous silica, and reacted at room temperature for a predetermined time (1 to 4 hours). In order to remove non-specifically adsorbed (HRP-antiBSA), this immobilized mesoporous silica was washed several times with pure water. Thereafter, freeze-drying was performed, and the dried sample was allowed to stand at 37 ° C. for an arbitrary time. Next, 400 μl of 50 mM Tris-HCl (pH = 7.5), 8 μl of a 5000 ppm phenol aqueous solution, and 2 μl of 30% aqueous hydrogen peroxide were added and reacted at 37 ° C. for 30 minutes. After centrifugation, 150 μl and 300 μl of 1% hexacyanoferrate and 1% 4-aminoantipyrine prepared with 150 μl of supernatant and 1 M glycine aqueous solution (pH = 9.6) were added and stirred. Thereafter, the absorbance near 500 nm was quickly measured, and the enzyme activity of HRP labeled with HRP-antiBSA specifically bound to the immobilized BSA was measured.

  In addition, non-specific ovalbumin (ovalbumin, 7.0 nm in diameter, IEP 4.9) was immobilized on mesoporous silica in the same manner as described above, reacted with the above-mentioned anti-BSA antibody, and the absorbance of both was measured. The difference was measured by the above procedure. As a result, non-specific ovalbumin had clearly lower HRP activity than the BSA-immobilized mesoporous silica according to this example. From these results, it was confirmed that BSA was immobilized on mesoporous silica and that an antigen-antibody reaction was effectively occurring in the pores after immobilization on mesoporous silica.

  Further, after the BSA-immobilized mesoporous silica obtained above and normal BSA powder were stored in a dry state at 40 ° C. for 3 weeks, the same change due to HRP activity was measured. As a result of setting the binding activity of both of them immediately before starting dry storage at 37 ° C. to 100, the non-immobilized BSA had a relative activity of 0 after 1 week and was completely inactivated. On the other hand, BSA immobilized on hierarchical mesoporous silica showed a relative activity of 90% or more even after 3 weeks.

  In this example, the surface of the short-axis oriented mesoporous silica synthesized in Example 1 was modified with a silane coupling agent, and α-amylase was immobilized on the silica surface by a covalent bond.

  1.0 g of the short-axis oriented mesoporous silica synthesized in Example 1 was added to 50 ml of a 10% (v / v) 3-aminopropyl triethoxysilane solution prepared with toluene, and the solution was added at 120 ° C. under a nitrogen atmosphere. Stir for 48 hours. After the reaction, the precipitate was filtered, washed with toluene, methanol, and dichloromethane, and then dried at room temperature.

  Next, 1.0 g of this dried sample was dissolved in 25 ml of a 2.5% glutaraldehyde solution prepared with a phosphate buffer solution (pH = 6.6) and stirred at room temperature for 1 hour. The obtained precipitate was washed four times or more with pure water and then dried at room temperature.

As a result of evaluating the short-axis oriented mesoporous silica modified with Glutaraldehyde by X-ray diffraction, the diffraction pattern was almost the same as that before modification, and it was confirmed that the mesopore structure was not broken. Moreover, as a result of identifying the functional group introduced into the silica surface using FT-IR, peaks due to R—CH═N, C═O, and —CHO were confirmed, and Si (CH ₂ ) was observed on the silica surface. _{3 N = CH (CH 2)} 3 CHO was confirmed to be covalently attached.

  Subsequently, α-amylase was immobilized in the pores of the modified mesoporous silica, and the stabilization effect was measured using a hydrolysis reaction of starch to maltose.

Α-Amylase was prepared to 1 mg / ml using 50 mM phosphate buffer (pH = 6.0), and 0.2 g of short-axis oriented mesoporous silica synthesized and modified by the above method was added to 1 ml of this solution. did. The mixed solution was impregnated using a shaker at 4 ° C. for 20 hours to adsorb α-amylase into the mesoporous silica pores. After completion of the reaction, the pellet was filtered and washed 3 times with pure water. At this time, each of the original enzyme solution and the supernatant was subjected to UV-Vis absorbance measurement. Using the absorption maximum of α-amylase at 280 nm, the amount of adsorption to mesoporous silica was calculated from the change in concentration before and after adsorption. The adsorbed α-amylase showed a high value of 140 mg / g. In the adsorption experiment of α-amylase under the same conditions to mesoporous silica not subjected to surface modification, the adsorption behavior was hardly exhibited. Therefore, α-amylase was bonded to —CHO modified on the silica surface and —NH _{2 of} α-amylase and immobilized on the silica surface.

  Subsequently, amylase-immobilized mesoporous silica and non-immobilized free amylase were each heat-treated in a sodium acetate buffer solution at 25 ° C. to 70 ° C., and then enzyme activity was measured.

  400 μl of 50 mM sodium acetate buffer (pH = 5.0) was added to 10 mg of the above amylase-immobilized mesoporous silica, and each was treated at 25 to 70 ° C. for 30 minutes. Centrifugation was performed after heating, and the amylase-immobilized silica was washed twice with pure water. Using the same 50 mM sodium acetate buffer, 0.125% soluble starch was prepared, 300 μl of this starch solution was added to the washed amylase-immobilized silica, and reacted at 40 ° C. for 15 minutes. After stopping the reaction, a supernatant was obtained by centrifugation, and 3 ml of 1 ml of 0.5N acetic acid and iodine-potassium solution (0.015% iodine-0.15% potassium iodide solution) was added to the supernatant solution and stirred well. Then, the absorption maximum at 700 nm was measured. Normal α-amylase had a relative activity of 20% under the conditions of 60 ° C. and 30 minutes, whereas α-amylase immobilized on short-axis oriented mesoporous silica had 90% under the same conditions. The above relative activity was shown and the stabilization effect was confirmed.

  A goat-derived IgG Fab fragment (diameter of about 12 nm) was immobilized in the pores of the short-axis oriented mesoporous silica synthesized in Example 1, and a specific binding reaction between an antigen and an antibody was used. This is an example of stability measurement.

  The short-axis oriented mesoporous silica synthesized in Example 1 and 10 mg of SBA-15 used in Comparative Example 1 were each prepared with a Fab fragment of anti-mouse immunoglobulin G (IgG) to 1 mg / ml with 10 mM phosphate buffer. 10 ml of solution was added. The solution was stirred at 4 ° C. for 6 hours to immobilize the antibody in the mesopores of mesoporous silica. The amount of Fab adsorbed was calculated by measuring the absorbance at 280 nm using a UV-Vis absorptiometer. In the short-axis oriented mesoporous silica, about 150 mg / ml Fab was observed in the pores. However, adsorption could not be confirmed at all with ordinary SBA-15.

  Then, it washed 3 times with pure water. A mouse-derived IgG antibody (abbreviated as HRP-IgG) labeled with horseradish peroxidase was added to the above antibody-immobilized mesoporous silica, and reacted at room temperature for a predetermined time (1 to 4 hours). In order to remove non-specifically adsorbed HRP-IgG, this antigen-antibody-immobilized mesoporous silica was washed several times with pure water. Thereafter, freeze-drying was performed, and the dried sample was allowed to stand at 37 ° C. for an arbitrary time. Next, 400 μl of 50 mM Tris-HCl (pH = 7.5), 8 μl of a 5000 ppm phenol aqueous solution, and 2 μl of 30% aqueous hydrogen peroxide were added and reacted at 37 ° C. for 30 minutes. After centrifugation, 150 μl and 300 μl of 1% hexacyanoferrate and 1% 4-aminoantipyrine prepared with 150 μl of supernatant and 1M glycine aqueous solution (pH = 9.6) were added and stirred. Thereafter, the absorbance at around 500 nm was quickly measured, and the enzymatic activity of HRP labeled on the IgG antibody specifically bound to the immobilized Fab fragment was measured.

  Further, non-anti-mouse IgG Fab fragments were immobilized on the short-axis oriented mesoporous silica in the same manner as described above. The difference in absorbance between the above-mentioned Fab and non-specific Fab was measured according to the above procedure. As a result, the non-anti-mouse IgG Fab fragment clearly had lower HRP activity than the anti-mouse IgG Fab fragment-immobilized mesoporous silica according to this example. From these results, it was confirmed that the active substance having a diameter of 12 nm was immobilized on the short-axis oriented mesoporous silica and that the antigen-antibody reaction was effectively carried out in the pores after the immobilization on the mesoporous silica.

  The Fab-immobilized mesoporous silica obtained above and the non-immobilized Fab powder were stored in a dry state at 37 ° C. for 3 weeks, and the change in HRP activity due to the antigen-antibody reaction was measured. As a result of setting the binding activity of both of them immediately before starting dry storage at 37 ° C. as 100, the normal Fab fragment had a relative activity of 0 in 12 hours and was completely inactivated. On the other hand, the antigen-antibody reaction by the Fab fragment immobilized on the short-axis oriented mesoporous silica showed a relative activity of 80% or more even after 2 weeks.

It is a schematic diagram for demonstrating the short axis orientation mesoporous material utilized for this invention. It is a schematic diagram of the structure of the stabilized enzyme using the short axis orientation porous material synthesize | combined in Example 1 of this invention. It is a result of the X-ray diffraction of the short axis orientation mesoporous material utilized for this invention. It is an image of the scanning electron micrograph of the short axis orientation mesoporous material utilized for this invention. 2 is a high-magnification image of a scanning electron micrograph of a short-axis oriented mesoporous material used in the present invention. FIG. 5 is a high-magnification image of a scanning electron micrograph in the cross section of the short-axis oriented mesoporous material shown in FIG. 4. It is a graph which shows the conversion rate and time passage when horseradish peroxidase is fix | immobilized in the short axis orientation mesoporous material synthesize | combined in Example 1, and an enzyme reaction is performed in toluene. It is a graph which shows the relationship between the relative activity of an enzyme, and the heat processing time when a horseradish peroxidase is fix | immobilized in the short axis orientation mesoporous material synthesize | combined in Example 1, and it heat-processes at 70 degreeC. It is a graph which shows the temperature dependence of relative activity at the time of fix | immobilizing a horseradish peroxidase in the short axis orientation mesoporous material synthesize | combined in Example 1, and heat-processing for 30 minutes at predetermined | prescribed temperature.

Explanation of symbols

11 Rod-like structure with mesopores 12 Mesopores 13 Pore walls 21 Short-axis oriented rod-like structures 22 Pore walls 23 Biological substances including enzymes and proteins 24 Specific binding to biological substances Fragment such as substrate

Claims (9)

Rod-shaped particles,
Having mesopores intersecting the rod-shaped major axis direction;
Particles characterized by carrying a biological substance in the mesopores.   The particle according to claim 1, wherein the length of the mesopore is greater than 10 times and not more than 30 times the pore diameter.   The particle according to claim 1, wherein the mesopore has a pore diameter of 10 nm or more and 30 nm or less.   The particle according to claim 1, wherein the particle has a plurality of mesopores, and the mesopores are arranged in a honeycomb shape.   The particle according to claim 1, wherein the biological substance is a protein.   The pore size distribution of the mesopores determined by nitrogen gas adsorption measurement has a single maximum value, and 60% or more of all mesopores are within the range of the pore size distribution width of 10 nm or less. Particles according to the first.   The particle according to claim 1, which has at least one diffraction peak in an angle region corresponding to a structural period of 1 nm or more in X-ray diffraction analysis. A sensor for detecting a specimen,
Particles according to claim 1;
With electrodes,
An electrical output signal based on a reaction between the specimen and the biological material carried in the mesopore is detected. In the specimen detection method,
Preparing a sensor using the particles of claim 1;
Applying a fluid containing a specimen to the sensor;
And a method of detecting an output signal based on a reaction between the biological material and the sample.

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